Reverse genetics reveals contrary effects of two Rdl-homologous GABA receptors of Helicoverpa armigera on the toxicity of cyclodiene insecticides

Abstract

The resistance to dieldrin gene (Rdl) encodes a subunit of the insect γ-amino butyric acid (GABA) receptor, and the encoded Rdl subunit is a major target site for cyclodiene and phenylpyrazole insecticides. Since the substitution of a single amino acid (Ala to Ser/Gly at position 302) of the Drosophila melanogaster Rdl gene was first identified to confer high level resistance to dieldrin, mutations at the equivalent positions have been reported to confer resistance to dieldrin and/or fipronil in a wide range of different insects. In the cotton bollworm Helicoverpa armigera, there are two Rdl homologs (HaRdl-1 and HaRdl-2) in close proximity on the Z chromosome, which as wild-type sequences, encode alanine and serine respectively at amino acid position 302. In the present study, we used the CRISPR/Cas9 gene editing approach to knock out HaRdl-1 and HaRdl-2 and establish two homozygous knockout strains (ΔRdl-1 and ΔRdl-2). The ΔRdl-1 strain showed low levels of resistance (8.0- to 9.3-fold) to three cyclodiene insecticides (endosulfan, aldrin and dieldrin) compared with the background SCD strain. In contrast, toxicity of the three cyclodiene insecticides to the ΔRdl-2 strain increased significantly (3.6- to 6.3-fold) when compared with the SCD strain. Genetic analysis indicated the obtained resistance to endosulfan and dieldrin in the ΔRdl-1 strain was sex-linked, which is consistent with the fact that HaRdl-1 locus is located on the Z chromosome. The above results demonstrate that both HaRdl-1 and HaRdl-2 are important determinants for the susceptibility of H. armigera SCD strain to the three cyclodiene insecticides, but have opposite effects. It was also found that HaRdl-1 and HaRdl-2 are involved, to some extent, in mediating sensitivity of H. armigera to avermectin and fipronil respectively. We speculate that the HaRdl-1 and HaRdl-2 subunits have different pharmacological properties, which contribute to the differential sensitivities of H. armigera to the tested cyclodienes and other insecticides.